The present invention relates to newly identified human deoxyribonuclease (DNase) protein, nucleic acid encoding such protein, the use of such protein and nucleic acid, as well as the production of such protein and nucleic acid, for example, by recombinant DNA methods.
Deoxyribonuclease (DNase) is a phosphodiesterase capable of hydrolyzing polydeoxyribonucleic acid, and is known to occur in several molecular forms. Based on their biochemical properties and enzymatic activities, DNase proteins have been classified as two types, DNase I and DNase II. DNase I proteins have a pH optimum near neutrality, an obligatory requirement for divalent cations, and produce 5xe2x80x2-phosphate nucleotides on hydrolysis of DNA. DNase II proteins exhibit an acid pH optimum, can be activated by divalent cations, and produce 3xe2x80x2-phosphate nucleotides on hydrolysis of DNA.
DNase from various species have been purified to a varying degree. For example, various forms of bovine DNase I have been purified and completely sequenced (Liao, et al., J. Biol. Chem. 248:1489-1495 (1973); Oefner, et al., J. Mol. Biol. 192:605-632 (1986); Lahm, et al., J. Mol. Biol. 221:645-667 (1991)), and DNA encoding bovine DNase I has been cloned and expressed (Worrall, et al., J. Biol. Chem 265:21889-21895 (1990)). Porcine and orcine DNase I proteins also have been purified and completely sequenced (Paudel, et al., J. Biol. Chem. 261:16006-16011 (1986); Paudel, et al., J. Biol. Chem. 261:16012-16017 (1986)).
DNA encoding a human DNase I has been isolated and sequenced and the DNA has been expressed in recombinant host cells, thereby enabling the production of human DNase I in commercially useful quantities. Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990). The term xe2x80x9chuman DNase Ixe2x80x9d will be used hereafter to refer to the mature polypeptide disclosed in Shak, et al.
DNA encoding other polypeptides having homology to human DNase I also have been identified. Rosen, et al., PCT Patent Publication No. WO 95/30428, published Nov. 16, 1995; Parrish, et al., Hum. Mol. Genet. 4:1557-1564 (1995).
DNase I has a number of known utilities and has been used for therapeutic purposes. Its principal therapeutic use has been to reduce the viscoelasticity of pulmonary secretions (mucus) in such diseases as pneumonia and cystic fibrosis (CF), thereby aiding in the clearing of respiratory airways. See e.g., Lourenco, et al., Arch. Intern. Med. 142:2299-2308 (1982); Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990); Hubbard, et al., New Engl. J. Med. 326:812-815 (1992); Fuchs, et al., New Engl. J. Med. 331:637-642 (1994); Bryson, et al., Drugs 48:894-906 (1994). Mucus also contributes to the morbidity of chronic bronchitis, asthmatic bronchitis, bronchiectasis, emphysema, acute and chronic sinusitis, and even the common cold.
The pulmonary secretions of persons having such diseases are complex materials, that include mucus glycoproteins, mucopolysaccharides, proteases, actin, and DNA. DNase I is effective in reducing the viscoelasticity of pulmonary secretions by hydrolyzing, or degrading, high-molecular-weight DNA that is present in such secretions. Shak, et al., Proc. Natl. Acad. Sci. 87:9188-9192 (1990); Aitken, et al., J. Am. Med. Assoc. 267:1947-1951 (1992). The DNA-hydrolytic activity of DNase I in pulmonary secretions may be reduced, however, as a result of the interaction of the DNase I with actin. Lazarides, et al., Proc. Natl. Acad. Sci. 71:4742-4746 (1974); Mannherz, et al., Eur. J. Biochem. 104:367-379 (1980). Accordingly, forms of DNase I that bind actin with lower affinity than human native DNase I, but that still possess DNA-hydrolytic activity should be useful therapeutic agents, especially in the treatment of patients having pulmonary secretions that comprise relatively large amounts of actin. Variants of human DNase I having reduced affinity for actin have been prepared synthetically and shown to be more potent than the native enzyme in reducing the viscosity of sputum of cystic fibrosis patients. Lazarus, et al., U.S. patent application Ser. No. 08/540,527 (filed Oct. 9, 1995).
The present invention provides a novel DNase, as well as analogs and variants thereof, that have DNA-hydrolytic activity but that are resistant to inhibition by actin. This novel polypeptide, referred to as LS-DNase, is of human origin.
The invention also provides nucleic acids encoding LS-DNase, recombinant vectors comprising such nucleic acids, recombinant host cells transformed with those nucleic acids or vectors, and processes for producing LS-DNase by means of recombinant DNA technology. The invention includes the use of such nucleic acids and vectors for in vivo or ex vivo gene therapy.
The invention also provides pharmaceutical compositions comprising LS-DNase, optionally together with a pharmaceutically acceptable excipient, as well as substantially purified antibodies that are capable of binding to LS-DNase.
The invention also provides methods for reducing the viscoelasticity or viscous consistency of DNA-containing material in a patient, comprising administering a therapeutically effective dose of LS-DNase to the patient. The invention is particularly directed to a method of treating a patient having a disease such as cystic fibrosis, chronic bronchitis, pneumonia, bronchiectasis, emphysema, asthma, or systemic lupus erythematosus, that comprises administering a therapeutically effective amount of LS-DNase to the patient. The invention also is directed to the use of LS-DNase in in vitro diagnostic assays of a viscous material (e.g., sputum) from a patient.
These and other aspects of the invention will be apparent to the ordinary skilled artisan upon consideration of the following detailed description.